JOURNAL OF BACTERIOLOGY, Dec. 1998, p. 6187–6192
0021-9193/98/$04.0010
Copyright © 1998, American Society for Microbiology. All Rights Reserved.
Vol. 180, No. 23
Rpb4, a Subunit of RNA Polymerase II, Enables the Enzyme
To Transcribe at Temperature Extremes In Vitro
SONIA ROSENHECK
AND
MORDECHAI CHODER*
Department of Molecular Microbiology and Biotechnology, Faculty of Life Sciences,
Tel-Aviv University, Tel-Aviv 69978, Israel
Received 9 July 1998/Accepted 28 September 1998
Rpb4 is a subunit of Saccharomyces cerevisiae RNA polymerase II (Pol II). It associates with the polymerase
preferentially in stationary phase and is essential for some stress responses. Using the promoter-independent
initiation and chain elongation assay, we monitored Pol II enzymatic activity in cell extracts. We show here that
Rpb4 is required for the polymerase activity at temperature extremes (10 and 35°C). In contrast, at moderate
temperature (23°C) Pol II activity is independent of Rpb4. These results are consistent with the role previously
attributed to Rpb4 as a subunit whose association with Pol II helps Pol II to transcribe during extreme
temperatures. The enzymatic inactivation of Pol II lacking Rpb4 at the nonoptimal temperature was prevented
by the addition of recombinant Rpb4 produced in Escherichia coli prior to the in vitro reaction assay. This
finding suggests that modification of Rpb4 is not required for its functional association with the other Pol II
subunits. Sucrose gradient and immunoprecipitation experiments demonstrated that Rpb4 is present in the
cell in excess over the Pol II complex during all growth phases. Nevertheless, the rescue of Pol II activity at the
nonoptimal temperature by Rpb4 is possible only when cell extracts are obtained from postlogarithmic cells,
not from logarithmically growing cells. This result suggests that Pol II molecules should be modified in order
to recruit Rpb4; the portion of the modified Pol II molecules is small during logarithmic phase and becomes
predominant in stationary phase.
the diauxic shift, cells continue to grow for one to three generations at a slower, albeit exponential, rate. Cells lacking
RPB4 grow more slowly than wild-type cells during the second
growth phase, exhibit a substantial decline in mRNA synthesis
relative to wild-type cells, do not enter stationary phase normally, and rapidly lose viability during starvation (5). Interestingly, the normal level of Rpb4 limits growth rate after but not
before the diauxic shift. Thus, whereas cells overexpressing
RPB4 grow indistinguishably from wild-type cells during log
phase, they grow substantially faster than wild-type cells during
post-diauxic shift growth phase (4).
The pattern of RPB4 expression differs from the pattern of
expression of the other Pol II subunit genes. Whereas mRNA
and protein levels of other subunits decline following the shift
from log to post-log phases, RPB4 mRNA and protein remain
constitutively high (3–5). Furthermore, in stationary phase, but
not during optimal growth conditions, Rpb4 protein level is
regulated posttranscriptionally. Thus, under optimal growth
conditions, when Rpb4 is dispensable, the Rpb4 protein level is
directly proportional to the RPB4 mRNA level. However, in
stationary phase, when Rpb4 is essential for maintaining viability, Rpb4 protein level is little affected by artificial changes
in its mRNA level (4). Taken together, the unusual phenotype
of rpb42 cells and the pattern of RPB4 expression indicate that
Rpb4 plays a vital role specifically during some stress conditions.
Rpb4 is known to interact with an essential Pol II subunit,
Rpb7 (6, 10, 11, 18). The association of Rpb7 with Pol II is
influenced by Rpb4, and it seems that they both interact with
the polymerase as a heterodimer, called Rpb4/7. However, the
functions of these subunits are not necessarily coupled under
all circumstances. First, RPB7 but not RPB4 is essential for
viability (18), indicating that Rpb7 can function in the absence
of Rpb4. Second, overexpression of RPB7 but not RPB4 can
influence cell morphology and induce pseudohyphal growth
(10). Furthermore, deletion of RPB4 has no effect on pseudohy-
Although changes in transcription are a hallmark of stress
responses (12), little is known about the mechanisms that permit the transcription apparatus itself to tolerate stress. Several
observations have led us to investigate the possibility that
Rpb4, a yeast RNA polymerase II (Pol II) subunit, plays a
critical role in enabling Pol II to transcribe during some stress
conditions. The yeast Saccharomyces cerevisiae Pol II is composed of 12 subunits (18). Rpb4 exhibits some unique features
distinguishing it from the other subunits. As for Rpb7 (15a) but
unlike the case for other subunits, the stoichiometry of Rpb4 is
dependent on growth conditions. In optimally growing cells,
the fraction of Pol II molecules containing Rpb4 is about 20%
(5, 11), and it gradually increases following the shift to postlogarithmic phases. Thus, in stationary phase virtually all Pol II
molecules contain Rpb4 (5), and these molecules, unlike Pol II
molecules obtained from logarithmically growing cells, can
form high-quality two-dimensional crystals (2, 8). RPB4 is not
essential for cell viability (17). Under optimal growth conditions at moderate temperatures (18 to 22°C), cells lacking
RPB4 (designated herein rpb42 cells) grow indistinguishably
from their wild-type counterparts (5). Consistently, under
these conditions, the global transcriptional activity in rpb42
cells is comparable to that in the wild-type strains. However
rpb42 cells rapidly lose the capacity for efficient growth and
global transcription as they experience higher or lower temperatures. In addition to the requirement for Rpb4 at temperature extremes, this subunit is required for efficient transcription in post-logarithmic phases (at moderate temperatures).
Normally, as yeast cells sense that nutrients are being depleted,
they alter their pattern of gene expression and briefly stop
growth; this event is termed the diauxic shift (9, 14). Following
* Corresponding author. Mailing address: Department of Molecular
Microbiology and Biotechnology, Faculty of Life Sciences. Tel-Aviv
University, Tel-Aviv 69978, Israel. Phone: (972) 36409030. Fax: (972)
36409407. E-mail: [email protected].
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J. BACTERIOL.
FIG. 1. Rpb4 is required for Pol II activity at high and low, but not moderate, temperatures. Wild-type and rpb42 cells were grown in rich medium (YPD) at 26°C
to stationary phase. Cell extracts were obtained as described in Materials and Methods. Pol II activity was tested at the indicated temperatures in the promoterindependent assay (see Materials and Methods). Rpb41 and Rpb42 represent incorporation kinetics for extracts from wild-type and rpb42 cells, respectively. The
reactions at 25 and 35°C (B and C) were done in the same experimental setup. The reactions at 10°C (A) were done in a different experiment, and therefore the extent
of incorporation should not be compared to those in panels B and C. All reactions were done at least three times with at least three different batches of cell extracts.
The extent of incorporation varied between the batches. However, the relative kinetics of wild-type Pol II versus pol IID4 at each specific temperature was highly
reproducible.
phal growth (6a). It is also worth noting that the human homolog
of Rpb7 can form a stable homodimer (1) and may interact as a
homodimer with Pol II independently of Rpb4. Taken together,
the results suggest that Rpb4 and Rpb7 have distinct functions
independent of each other, in addition to their role as a heterodimer.
Here we show that the interaction of Rpb4 with Pol II in
vitro permits the enzyme to transcribe at temperature extremes. Results presented here are consistent with the transcriptional phenotype of cells lacking RPB4 and support the
role suggested for Rpb4 as a Pol II subunit essential for transcription under nonoptimal temperatures.
MATERIALS AND METHODS
Yeast strains and medium. The wild-type strain SUB62 and its isogenic rpb4D1
(designated here rpb42) strain (MC11-1) were described previously (5). Z277,
carrying epitope-tagged RPB3, was described previously (11). RS420 (MATa
ura3-53 his4 trp1 leu2-3,112 rpb1-1) was a generous gift from R. Sternglanz. Cells
were grown in a YPD medium (2% Bacto Peptone, 1% yeast extract [Difco
Laboratories], 2% dextrose) at 25°C. For all experiments, the inoculum came
from cell cultures that had been growing in log phase for at least seven generations.
Antibodies and Western analysis. Affinity-purified anti-Rpb4 and anti-Rpb2
antibodies were a generous gift from A. Sentenac (7). The anti-Rpb1 C-terminal
domain monoclonal antibody 8WG16 was a generous gift from Nancy Thompson
and Richard Burgess (16). Western analysis was done as described previously (5).
Protein extraction. Whole-cell protein extraction was done essentially as described elsewhere (5). Briefly, proteins were extracted from 1 3 109 to 3 3 109
cells in 0.5 ml of PEB (50 mM Tris HCl [pH 7.9], 10 mM MgCl2, 0.3 M
ammonium sulfate, 1 mM dithiothreitol, 0.5 mM EDTA, 10% glycerol, 20 mg
each of aprotinin, antipain, and leupeptin per ml, 1 mM each phenylmethylsulfonyl fluoride and pepstatin A, 50 mg of Na-p-tosyl-L-lysine chloromethyl ketone
[TLCK] per ml [all protein inhibitors from Sigma]), using the glass beads (350 ml)
procedure. Protein concentrations determined by the Bradford assay (Bio-Rad)
were 3 to 11 mg/ml.
Transcription assay. Nonspecific initiation and chain elongation with poly(rC)
as the template was assayed as described elsewhere (15). After preincubation of
cell extracts at the desired temperature for 3 min, prewarmed transcription
mixture (15) was added and mixed by pipetting up and down while the tube was
in the water bath. To keep the reaction temperature constant, tubes were not
removed from the water bath during any manipulations. Immediately following
addition of the transcription mixture, one half of the reaction mixture was
transferred into a prewarmed tube containing a-amanitin (Sigma). The drug
(final concentration, 50 mg/ml) was mixed by pipetting up and down while the
tube was in the water bath. To terminate transcription, samples were spotted
onto 3MM paper which had been soaked in 10 mM EDTA and then air dried.
The 3MM paper containing all samples was subjected to trichloroacetic acid
(TCA) precipitation on ice as follows: 30 min of incubation in 0.2 M Na4P2O7–
10% TCA and three washes for several hours in 0.1 M Na4P2O7–5% TCA,
followed by washes with ethanol and then acetone. The paper was dried, and
radioactivity was measured in a scintillation counter. Pol II-specific incorporation
is calculated as the difference between the TCA-incorporated radioactivity (catalyzed by Pol I, Pol II, and Pol III) and that obtained in the parallel reaction
containing a-amanitin (catalyzed by Pol I and Pol III).
RESULTS AND DISCUSSION
Rpb4 is required for efficient transcription by Pol II only at
temperature extremes. Previously it was shown that RPB4 is
required for growth and efficient transcription at temperature
extremes in vivo (5, 17). In contrast, under optimal growth
conditions at moderate temperatures, Rpb4 was shown to be
dispensable for Pol II activity, as cells lacking RPB4 were able
to grow indistinguishably from wild-type cells under these conditions (5). These results raised two alternative but not mutually exclusive explanations, for the function of Rpb4. (i) Rpb4
affects the expression of a subpopulation of genes (e.g., some
specific heat shock genes) needed for transcription of other
genes under some stress conditions, and (ii) Rpb4 affects the
enzymatic activity of pol II in a direct and promoter-independent manner. According to the latter possibility, the involvement of Rpb4 is dispensable for Pol II activity under optimal
conditions but essential for its enzymatic activity under some
nonoptimal conditions. To test the hypothesis that Rpb4 is
directly required for Pol II activity at temperature extremes, we
monitored Pol II activity in extracts from RPB41 and rpb42
cells at various temperatures. To monitor Pol II activity per se,
independently of other factors, we used the promoter-independent initiation and chain elongation assay developed by Ruet
et al. (15). It was previously shown that the removal of Rpb4
had little effect on Pol II activity at moderate temperatures in
this assay (6). Results shown in Fig. 1B led to the same conclusion. Rpb4 has little effect on Pol II activity within a temperature window ranging between 20 and 28°C (Fig. 1B and
results not shown). In contrast with the activity at moderate
temperatures, Rpb4 is shown here to be required for efficient
activity of Pol II at high or low temperatures. Figure 1A demonstrates that extract from RPB41 cells can support efficient
Pol II transcription at 10°C, whereas Pol II activity in extract
from rpb42 cells is severely impaired at this temperature. Similarly, extract from RPB41 cells can support efficient Pol II
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Rpb4-DEPENDENT Pol II TRANSCRIPTION AT TEMPERATURE EXTREMES
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FIG. 2. Rescue of Pol II activity at high temperature is dependent on the growth phase of the cells used to extract the proteins. Wild-type (RPB41) and rpb42 cells
were grown in rich medium (YPD) at 26°C. Equal amounts of cells were harvested at various growth phases. Cell extracts were prepared and Pol II activity was assayed
as for Fig. 1. A. Incorporation kinetics for extracts prepared from RPB41 cells harvested at early log phase was determined at 24 and 35°C. (B) Heat resistance of Pol
II as a function of growth phase of the cells used as a source of protein extract. Heat resistance is defined as the ratio between the Pol II-specific radioactivity
incorporated at 35°C during 30 min and that incorporated at 25°C during 30 min. EL, early log; LL, late log; DS, diauxic shift; SG, slow-growth phase; SP0, beginning
of stationary phase; SP3, 3 days into stationary phase.
transcription at 35°C, whereas Pol II activity in extract from
rpb42 cells is severely impaired at this temperature. The enzyme lacking Rpb4 exhibits some initial activity at 35°C and
only after a few minutes loses this activity completely. This late
inactivation suggests that in vitro, some early stages of the
initiation process are less dependent on Rpb4 at this nonoptimal temperature. At 10°C, the Pol II extracted from rbp42 cells
(Pol IID4) is so poorly active that this late effect can hardly be
observed. These results are in agreement with the in vivo
results, favoring our suggestion that Rpb4 is essential for the
appropriate function of Pol II under temperature stresses.
Experiments described in Fig. 1 were carried out with extracts from stationary-phase cells, during which Pol II molecules carry stoichiometric amounts of Rpb4 (5). Therefore, the
effect of Rpb4 on enzymatic activity could easily be tested.
Figure 2A shows that Pol II activity in extract from logarithmically growing wild-type cells, in which most Pol II molecules
do not contain Rpb4 (5), is heat sensitive. Figure 2B shows that
the shift from heat-sensitive to heat-tolerant Pol II occurs
following the shift from logarithmic to post-logarithmic phases.
Specifically, Pol II extracted from optimally and logarithmically growing cells exhibited heat sensitivity similarly to the Pol
II extracted from rpb42 cells (Fig. 2B; compare columns EL
for RPB41 and rpb42); however, Pol II extracted from postlogarithmic wild-type (but not rpb42) cells became heat tolerant. During the diauxic shift, the transition phase between
logarithmic and slow growth (see the introduction), Pol II was
still heat sensitive. These results are in good correlation with
the stoichiometry of Rpb4 found previously in vivo. In logarithmic phase and during the diauxic shift, most Pol II molecules do not contain Rpb4 (5) and the in vitro activity of Pol II
is heat sensitive (Fig. 2A). Following the diauxic shift, most Pol
II molecules contain Rpb4 (5) and the in vitro activity of Pol II
is heat tolerant (Fig. 1C and 2B). Interestingly, Pol II extracted
from cells which had been growing logarithmically at 37°C
carry substoichiometric amounts of Rpb4 (5). Consistently, the
in vitro activity of this Pol II molecules is heat sensitive (results
not shown).
Rpb4 is present in excess over Rpb1, Rpb2, and Rpb3. Previous results from in vivo experiments and the present results
obtained in vitro (Fig. 2) demonstrate that interaction of Rpb4
with the Pol II complex is influenced by nutritional conditions.
We sought to identify what determines the interaction between
Rpb4 and the other Pol II subunits. First, we examined
whether Rpb4 level is a limiting factor by performing two sets
of experiments. Results of an immunoprecipitation experiment
using antibodies directed against the C-terminal domain of
Rpb1 (Fig. 3A) demonstrated that most Rpb4 molecules did
not precipitate with the Pol II complex whereas Rpb1 and
Rpb2 subunits precipitated quite efficiently (compare the levels of individual subunits in lanes P and S). Thus, whereas
Rpb4 is much higher in lanes S than in lanes P, the inverse is
observed for Rpb1 and Rpb2. This differential immunoprecipitation was observed in extracts from both log-phase and stationary-phase cells (Fig. 3A). Sedimentation through a sucrose
gradient (Fig. 3B) revealed that most Rpb4 molecules do not
cosediment with the Pol II complex and Pol II activity (fractions 5 to 7) but instead sediment more slowly (fractions 7 to
12). This result indicates that most Rpb4 molecules do not
associate in a stable complex with Pol II. Excess of free Rpb4
over Pol II-associated Rpb4 is observed by sedimenting extracts from both logarithmically growing (Fig. 3B) and from
stationary-phase (results not shown) cells. Taken together,
these observations demonstrate that Rpb4 is in excess over
Rpb1, Rpb2, and Rpb3, suggesting that Rpb4 is not a limiting
factor. Thus, changes in Rpb4 level are not the main determinants of the extent of Rpb4 interaction with the Pol II complex.
Indeed, the Rpb4 level does not increase following the shift
from log to post-log phase but remains close to a constant
value during all growth phases (4). These results focused our
attention to the posttranslational modifications of either Rpb4
or other Pol II subunits as the cause for the differential inter-
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ROSENHECK AND CHODER
FIG. 3. Pol II-free Rpb4 is present in excess over Pol II-associated Rpb4. (A)
Immunoprecipitation experiment. Pol II was immunoprecipitated from extract
obtained from logarithmically growing cells (Log) or from stationary-phase cells
(SP). Immunoprecipitation, using a monoclonal antibody against the C-terminal
domain of Rpb1 (8WG16), was carried out as described previously (5). Following
immunoprecipitation, both the immunoprecipitated material (lanes P) and one
half of the unprecipitated supernatant (lanes S) were electrophoresed, then
electrotransferred onto a nitrocellulose filter, and probed with antibodies against
the indicated Pol II subunits (see Materials and Methods). Antibody 8WG16 was
used to detect Rpb1, affinity-purified rabbit anti-Rpb2 polyclonal antibodies
were used to detect Rpb2, and affinity-purified rabbit anti-Rpb4 polyclonal
antibodies were used to detect Rpb4. (B) Sucrose gradient. Protein extract (270
mg) obtained from Z277, a strain carrying hemagglutinin epitope-tagged Rpb3
(11), was sedimented through a 5 to 20% (wt/wt) sucrose gradient, using an
SW60 rotor at 60,000 rpm (485,000 3 g) for 2.5 h at 4°C. Gradient was fractionated into 12 fractions, and Pol II activity in 9 fractions was monitored at 24°C as
described in Materials and Methods (upper panel). Fifty microliters from each
fraction was added to 13 Laemli sample buffer and boiled for 3 min, and samples
were electrophoresed. Following electrophoresis, proteins were electrotransferred onto nitrocellulose filters and probed with antibodies against the indicated
subunits as described previously (5). Rpb1, Rpb2, and Rpb4 were detected by the
antibodies used for panel A. To detect the epitope-tagged-Rpb3, antibody
12CA5 was used. Lane M, purified Pol II (carrying wild-type Rpb3, which is not
detected by antibody 12CA5).
J. BACTERIOL.
action of Rpb4 with the Pol II complex in log and stationary
phases.
Growth phase-dependent modification of the remainder of
Pol II is required for its interaction with Rpb4. To determine
whether Rpb4 must be modified to functionally interact with
the Pol II complex, we examined the possibility that recombinant Rpb4, produced in E. coli, can rescue the activity of Pol
IID4 at high temperature. Results in Fig. 4A demonstrate that
the addition of a recombinant Rpb4 prior to transcription
initiation restored full activity of Pol IID4 at high temperature.
The results suggest that eukaryotic cell-specific posttranslational modification is not required for the functional interaction of Rpb4 with the Pol II complex. To determine whether
Pol II subunits other than Rpb4 must be modified to interact
with Rpb4, we carried out mixing experiments in which extracts
from rpb42 cells were mixed with extracts from rpb1-1 cells
which had been heated at 42°C for 18 min. Pol II in a prewarmed extract from the rpb1-1 strain is completely inactive,
due to a defect in its Rpb1 (6, 13) (Fig. 4B). As shown in Fig.
1C, 2B, and 4A, Pol II in the rpb4D extract is inactive at 35°C
due to the absence of Rpb4. Thus, each extract alone cannot
support efficient RNA synthesis in the promoter-independent
assay at 35°C. Activity at 35°C can be restored if the Rpb4,
present in excess over Pol II complex in the extract from the
rpb1-1 strain, can functionally interact with the Pol IID4 extracted from the rpb4D1 cells. Note that in these mixing experiments the main source of Rpb4 is the unbound (Pol II-free)
Rpb4 which is present in a large excess over the Pol II-associated Rpb4 both during the logarithmic growth phase and in
stationary phase (Fig. 3). In the mixing experiments, Rpb4 was
diluted only twofold, as equal volumes of extracts were mixed,
and it remained in excess over Pol II complexes. As shown in
Fig. 4B, when preheated rpb1-1 extract was mixed with extract
from logarithmically growing rpb42 cells, no complementation
could be observed; Pol II was active at 25°C but inactive at
35°C. Thus, although unbound Rpb4 molecules were present
in excess over Pol II complexes, they could not interact with
Pol II and restore its activity at 35°C. However, when preheated extract from rpb1-1 cells was mixed with an extract from
stationary rpb42 cells, Pol II regained activity at the high temperature. Thus, Rpb4 could interact with Pol II present in the
stationary-phase extract but not with Pol II present in the
log-phase extract. The possibility that the largest subunit can
change from Pol IID4 to the heat-inactivated polymerase is
unlikely; dissociation of this subunit from the polymerase under transcription conditions has never been observed, nor has
dissociation of the homologous subunit from the E. coli enzyme (6). To summarize, results in Fig. 4 demonstrate that Pol
II extracted from stationary cells can be rescued from heat
inactivation by Rpb4, whereas Pol II extracted from logarithmically growing cells cannot. The source of Rpb4, whether E.
coli (Fig. 4A), or log-phase (Fig. 4B) or stationary (data not
shown) yeast cells, is not important for its ability to render Pol
II heat resistant. Therefore, we suggest that modification of the
Pol II complex, not Rpb4, is required to recruit Rpb4 and that
this modification occurs predominantly following the shift from
log to post-log phases. The identification of the Pol II subunit(s) that becomes modified and the nature of this modification remain to be determined.
Does the ability of Rpb4 to render Pol II resistant to temperature extremes in vitro correlate with its known function in
vivo? We summarize below our present results and previous
results obtained in vivo (3, 4). First, under optimal growth
conditions at moderate temperatures (18 to 23°C), cells lacking
RPB4 can grow and transcribe genes almost indistinguishably
from their wild-type counterparts. Similarly, in the test tube at
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Rpb4-DEPENDENT Pol II TRANSCRIPTION AT TEMPERATURE EXTREMES
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FIG. 4. Activity at high temperature of Pol IID4 extracted from post-log-phase but not from logarithmically growing cells can be rescued by Rpb4. (A)
Reconstitution of Pol IID4 activity at high temperature with recombinant Rpb4 produced in E. coli. Rpb4-GST fusion protein was expressed in E. coli by using plasmid
pGEX-2T (Pharmacia) followed by purification on a glutathione-Sepharose column, as instructed by the manufacturer. The fusion protein was cleaved with thrombin,
and the release of free Rpb4 was ascertained by Western analysis (not shown). Reactions were carried out at 35°C as described in Materials and Methods. Pol
IID41Rpb4 (squares), the thrombin digest (0.5 mg) was preincubated with 33 mg of extract from stationary rpb42 cells at 30°C for 15 min followed by 10 min at 25°C
and 1 min at 35°C before the reaction commenced; Pol IID41GST (triangles), 33 mg of extract from stationary rpb42 cells preincubated with 0.5 mg of GST as described
above; Pol II WT (circles), 33 mg of extract from stationary wild-type (RPB41) cells. (B) In vitro complementation between rpb42 and heat-treated rpb1-1 extracts. Cell
were grown at 25°C in YPD and harvested at the indicated growth phase, and their proteins were extracted, as described in Materials and Methods. Extracts were
prepared from logarithmically growing (L) rpb1-1 cells or from logarithmically growing or stationary (S) rpb42 cells, and the protein concentration in each extract was
brought to 2.5 mg/ml in PEB (see Materials and Methods). The rpb1-1 extract was preheated at 42°C for 18 min to inactivate Pol II. Equal volumes of extracts,
containing equal amounts of protein, were mixed at various combinations, specified below the columns, before transcription reactions were initiated. In the reactions
containing only one extract, an equal volume of PEB was added (-). Note that in these mixtures the concentration of the bulk protein is lower than that in the other
mixtures. However, preliminary experiments demonstrated that this difference had no significant effect on Pol II activity. The various mixtures were incubated at 30°C
for 15 min, then at 23°C for 45 min, and finally for at 38°C for 1 min. After cooling in ice, each mixture was divided into two equal samples and transcription was assayed
at 24 or 35°C as described in Materials and Methods. Relative Pol II activity was calculated with respect to the activity in extract from the logarithmically growing rpb42
cells at 24°C (defined arbitrarily as 1).
moderate temperatures, Pol II lacking Rpb4 is as active as Pol
II containing Rpb4. Second, rpb42 cells cannot grow at temperature extremes (below 13°C and above 32°C); furthermore,
when these mutant cells are shifted from moderate to high
temperature, their Pol II activity is rapidly lost. Similarly, in the
test tube, Pol II requires Rpb4 for its activity at temperature
extremes. Third, in log phase only a minor subpopulation of
Pol II molecules contain Rpb4 (stoichiometry of ;0.2). Consistently, Pol II molecules extracted from logarithmically growing wild-type cells are heat sensitive, as are those extracted
from rpb42 cells. Fourth, in stationary phase, stoichiometric
amounts of Rpb4 are found associated with the Pol II. Consistently, Pol II molecules extracted from stationary-phase cells
are heat resistant. Thus, results obtained in vitro and described
in this paper are in accord with results found previously in vivo
and are likely to be biologically relevant.
The specific requirement for Rpb4 at temperature extremes
shown in this study favors a model in which a major, but not
necessarily the sole, role of this subunit is to permit the enzyme
to function under nonoptimal conditions. It is worth noting
that the requirement for Rpb4 at temperature extremes can
potentially be an indirect effect. For example, it is possible that
Rpb4 is required for recruiting yet another factor (e.g., Rpb7)
which helps Pol II to transcribe at temperature extremes.
Rpb4/7 was suggested to play a role other than those related
directly to the transcriptional response to stress. Pol II lacking
Rpb4/7 was shown to be deficient in selective transcription
initiation in vitro (6). Recently, electron crystallography of Pol
II molecules extracted from stationary-phase cells revealed
that the molecules containing Rpb4/7 differ in conformation
from those lacking Rpb4/7 (2, 8). These results were interpreted in terms of open and closed conformations that the
enzyme undergoes during the initial phases of transcription
initiation process. Jensen et al. (8) have also proposed that
Rpb4/7 heterodimer stabilizes the paused Pol II located downstream of heat shock promoters. Our results, demonstrating
that Rpb4 is required for Pol II activity at temperature extremes, support a model in which Rpb4 plays a global role
during stress, in a promoter-independent fashion, rather than
a specific role restricted to the transcription of heat shock
genes (see first paragraph in Results). It is possible, then, that
the addition of Rpb4/7 and the specific conformational change
that it elicits (2, 8) become critical for the global Pol II activity
especially during some stress conditions. Alternatively, it is
possible that the biochemical process leading to this conformational change is important under all conditions. Yet, during
some nonstress conditions it can be carried out by a factor
other than Rpb4, and only during stress is Rpb4 irreplaceable.
ACKNOWLEDGMENTS
We thank A. Sentenac and N. Thompson for antibodies, D. Chau for
purified Rpb4, N. Woychick for the disruption plasmid, R. Sternglanz
for strain RS420, and A. Krauskoff for critically reading the manuscript.
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ROSENHECK AND CHODER
This work was supported by the Israel Science Foundation founded
by the Israel Academy of Sciences and Humanities to M.C.
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